Serum-free stable transfection and production of recombinant human proteins in human cell lines

ABSTRACT

The present invention relates to an improved method for the serum-free production of an immortalized human cell line stably transfected under serum-free conditions with a specific vector carrying the gene coding for the protein of interest. Furthermore the invention relates to a production cell line obtained by said method, a production method for said protein of interest utilizing said production cell line, and the specific vector carrying the gene of interest itself.

Applicants incorporate by reference the material contained in the accompanying computer readable Sequence Listing identified as SEQ_ST25.txt, having a file creation date of Dec. 19, 2007 12:27:10 P.M. and a file size of 192,651 bytes.

The present invention relates to an improved method for the serum-free production of an immortalized human cell line stably transfected under serum-free conditions with a specific vector carrying the gene coding for the protein of interest. Furthermore the invention relates to a production cell line obtained by said method, a production method for said protein of interest utilizing said production cell line, and the specific vector carrying the gene of interest itself.

BACKGROUND

The recombinant production of human proteins is generally performed by cultivation of stably transfected eukaryotic and preferably mammalian cell lines and isolation of the protein from the culture broth. In case the recombinant proteins are intended for pharmaceutical applications, it was for a long time general practice to employ non-human cell lines in order to exclude the risk of copurifying infectious agents which may be harbored and expressed by human cells.

In the production of some human proteins, such as human blood clotting factor VIII, the use of non-human cell lines were found to entail certain disadvantages, e.g. unsatisfactory secretion levels of the expressed protein into the medium. It is believed that this may be due to slight differences within different types of mammalian cells concerning intracellular pathways for protein translation and modification, which also might have an effect on the biological activity of the expressed polypeptide. Apart from this, there were concerns that therapeutic proteins purified from non-human expression systems are contaminated with cellular components which can give rise to antigenic reactions in the patients. Also a concern was the non-human glycosylation pattern found on human proteins recombinantly produced in non-human expression systems. It is thought that this increases the likelihood of antigenic reactions in the patient. Furthermore, biological stability and efficacy of blood proteins such as clotting factors is substantially influenced by their N-glycosylation pattern. Especially peripheral and terminal monosaccharides are important, because they are detected by specific receptors from cells which are responsible for their degradation. Clotting factors, for example, carry sialic acid residues as terminal monosaccharides. Modification on the composition of sialic acids in the antennae of glycoproteins can result in heterogenous glycosylation patterns. Thus, biological stability and efficacy is crucially involved when modification occurs. Hence, it is an important consideration in the production of recombinant clotting factors to evaluate the influence of glycosylation from non-human production cell lines versus human cell lines.

On the other hand, general methods for high level protein expression of a desired gene comprising immortalized, stably transfected mammalian cell lines expressing viral transcription activator proteins were made available (e.g. U.S. Pat. No. 5,712,119). These cell lines can be transformed with a vector construct where a suitable viral transcription promoter is operatively associated with a DNA sequence defining a gene of interest, the transcription activator proteins provided by the cell lines activate the viral transcription promoter and hence initiate the expression of the gene of interest.

As important as the cell line is the vector used for the introduction of the recombinant gene into an immobilized production cell line. A wide variety of vectors were utilized for translation of mammalian proteins, (for example, Witsch-Baumgartner, M et al. Am. J. Genet (2000). 66, 402-412 cloned DHCR7 cDNA into pCI-neo mammalian expression vector and expressed in the HEK 293 cells; McGarvey, T. W. et. al. Oncogene (2001) 20, 1041-1051 cloned TERE1 gene into the pTARGET mammalian expression vector and expressed in the human bladder transitional cell carcinomas; and Lin Lin et. al. J Biol Chem (2002) 277 (44) 41872-8 cloned the AchR gene into mammalian cell expression vector pEF6/myc-His vector and expressed it in 293 cells). A recently developed very potent vector which has proven to be capable of over-expression of recombinant proteins is the so-called pcDNA™ 3.1 vector of Invitrogen. Li J. et al., Life Sci. 2004 Apr. 16; 74(22):2693-705 have successfully over-expressed histone deacetylases using pcDNA 3.1 in HEK 293 cells. The cells were stably transfected and cultured in the presence of serum. Yuan-Gen Fu. et al., World J Gastroenterol 2003 have produced recombinant Caspase-3 using a pcDNA 3.1(+) based eukaryotic vector on gastric cancer cell line SGC7901 transiently transfected with said vector and cultured in the presence of serum. Ma H. et al., Invest Ophthalmol V is Sci. 2000 December; 41(13):4232-9 examined the lack of stable protein and loss of enzymatic activity expressing Lp82 and Lp82-related proteins subcloned into pcDNA3.1 vector using COS-7 as cell line. The cells were transiently transfected and cultured in the presence of serum in the medium. Thioredoxin overexpression prevents NO-induced reduction of NO synthase activity in lung endothelial cells. Zhang J. et al., Am J. Physiol. 1998 August; 275(2 Pt 1): L288-93 disclose the overexpression of thioredoxin gene in cultured porcine pulmonary artery endothelial cells by transient transfection of these cells with pcDNA 3.1 vector. The transfected cells were cultured in medium supplemented with serum. Shinki T. et al., Proc Natl. Acad. Sci. USA 1997 Nov. 25; 94(24):12920-5 compared a full length cDNA for the rat kidney mitochondrial cytrochrome P450 mixed function oxidase, 25-hydroxyvitamin D3-1alpha-hydroxylase with vitamin D-deficient rat kidney cDNA and subcloned it into mammalian expression vector pcDNA 3.1 (+) and transiently transfected the vector into COS-7 transformed monkey kidney cells. The transfected cells were cultured in medium supplemented with serum. Zhang et al., Acta Biochimica et Biophysica Sinica 2004, 36(10): 707-712 disclose the transfection of human embryonic kidney 293 cells with pcDNA containing a gene coding for the humanized 520C9 single chain Fv antibody/human interleukin-2 fusion protein. Supernatant was taken after having cultured the cells for three days in serum-free SFM II media. The resultant fusion protein possessed binding specificity against p185 (promising target for antibody therapy in breast cancer) and retained the important immuno-stimulatory activities of IL-2. Chen, J. Z. et al., Int J Biochem Cell Biol. 2004 August; 36(8):1554-61 over-expressed Bim proteins, which are essential factors for apoptosis, using HEK 293 cells transfected with pcDNA-Bim alpha3.

A further measure for increasing the safety of recombinant proteins for pharmaceutical applications is the use of serum-free medium in the culturing process, as the use of serum represents a safety hazard as well as a source of unwanted contaminations. Such serum-free cultivation has the drawback that the yields of the production process are generally significantly reduced. A further safety concern is the use of serum when transfecting the host cells as a regular way in the practice, as the use of serum in the transfection procedure may cause unwanted biological material to be integrated into the cells which later on contaminated the product expressed by the cells in the production process. While some of the available methods for the production of recombinant proteins (including those mentioned above) do allow serum-free cultivation, serum-free stable transfection of human cells is not known. In the 19^(th) ESACT Meeting, Harrogate, 5-8 Jun. 2005 the serum free transfection of CHO cells was suggested by Kuchenbecker et al. Thus, it is desirable to develop an effective and safe method to produce human recombinant proteins.

SUMMARY OF THE INVENTION

Surprisingly, it was found that a non-contaminated human protein (i.e. a protein preparation free of unwanted protein by-products) can be obtained in good yield from immortalized human cell lines stably transfected, under serum-free conditions, with the gene encoding the protein of interest. In more detail, the present invention provides:

-   -   (1) a method for preparing an immortalized human cell line         stably transfected with a nucleic acid sequence comprising a         gene encoding a human target protein or a derivative or mutant         thereof, a promoter and a bovine growth hormone polyadenylation         (polyA) signal, said promoter and polyA signal being linked to         the 5′ and 3′ end of the gene encoding said human target         protein, respectively, which method comprises transfecting an         immortalized human host cell line under serum-free conditions         with a transfection vector comprising said nucleic acid sequence         and an origin of replication;     -   (2) the method of (1) above wherein the transfection vector is         derived from pcDNA 3.1 vector having the sequence of SEQ ID NO:4         or 5;     -   (3) the method of (1) or (2) above, wherein the human cell line         is a human embryonic kidney cell selected from 293 cells (ATCC         CRL-1573; DSM ACC 305), FreeStyle 293 cells (hereinafter “293F”         cells; Invitrogen R79007), and 293T cells (ATCC CRL 11268; DSM         ACC 2494);     -   (4) the method of (1) to (3) above, wherein the human protein is         blood clotting factor IX, (e.g. as encoded by in bps 939 to 2324         of SEQ ID NO:1), alpha-1-antitrypsin (hereinafter “A1AT”; e.g.         as encoded by bps 913 to 2259 of SEQ ID NO:2), blood clotting         factor VIII (including wt factor VIII as shown in SEQ ID NO:8 or         a B-domain deleted factor VIII mutant as encoded by bps 783 to         5162 of SEQ ID NO:3), factor VII/VIIa (including the a and b         form thereof encoded by SEQ ID NOs:13 and 14), G-CSF (including         the G-CSF a, b and c form shown in SEQ ID NOs:15, 16 and 17,         respectively), or von Willebrand factor (vWF);     -   (5) a transfection vector comprising an origin of replication         and a gene encoding a human protein as defined in (1) and (2)         above, preferably said transfection vector being a pcDNA3.1         vector comprising the gene for a human protein as defined in (4)         above;     -   (6) an immortalized human cell line obtainable by the method as         defined in (1) to (5) above, preferably said human cell line         being as defined in (3) or (4) above; and     -   (7) a method for the recombinant production of a human target         protein or a derivative or mutant thereof which comprises         culturing an immortalized human cell line as defined in (6)         above, preferably under serum-free conditions.

DESCRIPTION OF THE FIGURES

FIG. 1: The wild-type human clotting factor IX (FIX) protein. A schematic drawing of the FIX gene with its 5′ untranslated (5′ UTR) region and its 3′ UTR region. The eight domains of the unprocessed 461 amino acid protein are indicated: S: signal peptide; P: propeptide; Gla domain: γ-carboxyglutamyl domain; H domain: hydrophobic sequence; EGF domain: epidermal growth factor-like domain; L: linking sequence; AP: activation peptide; Catalytic domain. The FIX mature protein has a length of 415 amino acids and an approximate molecular weight of 55 kDa.

FIG. 2: The vector pcDNA3.1-FIX. The circular DNA vector comprises 6,960 base pairs, the exact sequence thereof being given in SEQ ID NO:1. In the schematic drawing the CMV promoter (CMV), the human FIX gene (hFIX), the f1 origin (f1), the hygromycin (Hyg) gene under control of the SV40 promoter (SV40), a poly A region (SV40 poly A), the pUC origin and the ampicillin (Amp) resistance gene are indicated, as well as numerous restriction sites. This vector is derived from the resequenced pcDNA 3.1 vector pcDNA3.1Hygro(+)-zz of SEQ ID NO:5.

FIG. 3: The vector pcDNA3.1-A1AT. The circular DNA vector comprises 6,914 bps, the exact sequence thereof being given in SEQ ID NO:2. In the schematic drawing the CMV enhancer promoter, the A1AT cDNA, bovine growth hormone polyadenylation (polyA) signal including a transcription termination sequence for enhanced mRNA stability, the f1 origin (f1), the hygromycin (Hyg) gene under control of the SV40 promoter (SV40), the SV40 poly A region (SV40 poly A), the pUC origin and the ampicillin (Amp) resistance gene are indicated, as well as numerous restriction sites. This vector is derived from vector pcDNA3.1Hygro(+)-zz of SEQ ID NO:5.

FIG. 4: Transient transfection of different human embryonic kidney cells with vectors coding for alpha-1-antitrypsin. A cell line comparison in transient transfection studies is shown. The amount (%) of alpha-1-antitrypsin (A1AT) expressed per 10⁶ cells is shown for 293, 293T and 293F cells. The A1AT amount expressed in 293F cells transiently transfected with pcDNA3.1-A1AT has been set as 100%.

FIG. 5: Transient transfection of 293F cell line with different vectors coding for alpha-1-antitrypsin. A comparison of A1AT concentrations expressed from different vectors using transiently transfected freestyle 293F cell line is shown. The expression level of A1AT pcDNA3.1-A1AT has been set as 100%. Various other vectors carrying the A1AT gene were also tested: An in-house vector pTG1-A1AT (an in-house vector for producing human recombinant A1AT as shown in FIG. 10), the pCMV Script® A1AT (Stratagene) and pcl neo-A1AT (Promega) were compared against the pcDNA3.1-A1AT (pcDNA3.1). None of the other vectors came close to the high expression levels observed with pcDNA3.1-A1AT.

FIG. 6: SDS-PAGE and Western blot of cell culture supernatant. Aliquots of the supernatant of freestyle 293F cells transiently transfected with pcDNA3.1-A1AT (lanes 1-3 and 6-8) or with a GFP-expressing control plasmid (lanes 4 and 8) were analysed both by SDS-PAGE and Western blot. Lane 1 contains a size marker, and lane 5 is empty. The band for A1AT is marked with an arrow. Also visible is the 27 kDa band corresponding to GFP in lanes 4 and 8.

FIG. 7 shows the vector pcDNA 3.1-F.VIII. The vector comprises 9,975 bps, the exact sequence thereof being shown in SEQ ID NO:3. The factor VIII protein encoded by bps 783 to 5162 is a B-domain deleted factor VIII mutant as disclosed in WO 01/70968. Again this vector is derived from vector pcDNA 3.1 Hygro(+)-zz of SEQ ID NO:5.

FIG. 8: Comparison of the average amount of produced factor VIII of the best three stably transfected clones, transfecting 293 and 293F cells with pcDNA3.1-FVIII and the in-house vector pTGF8-2hyg-s, the exact sequence thereof being given in SEQ ID NO:7.

FIG. 9 shows the vector pTG1-A1AT. The vector comprises 5,610 bps, the exact sequence thereof being shown in SEQ ID NO:6.

FIG. 10 shows the vector pCR2.1d2-GCSFb. The vector comprises 4,542 bps, the exact sequence thereof being shown in SEQ ID NO:21.

FIG. 11 shows the vector pDNA3.1-GCSFb. The vector comprises 6,237 bps, the exact sequence thereof being shown in SEQ ID NO:22.

FIG. 12 shows the vector pCINeo-GCSFb. The vector comprises 6,101 bps, the exact sequence thereof being shown in SEQ ID NO:23.

FIG. 13 shows the vector pCMVScript-GCSFb. The vector comprises 4,920 bps, the exact sequence thereof being shown in SEQ ID NO:24.

FIG. 14 shows the vector pTG2-GCSFb-hyg-as. The vector comprises 6,917 bps, the exact sequence thereof being shown in SEQ ID NO:25.

FIG. 15 compares the amount of rhG-CSF produced by different expression constructs according to Example 9.

FIG. 16 shows a western blot of rhG-CSF produced according to Example 9.

DETAILED DESCRIPTION OF INVENTION

The present invention provides an improved method for the transfection and production of human recombinant proteins in human immortalized cell lines completely under serum- and protein-free conditions. It allows the serum-free transfection and production of human proteins. The method may include one or more purification step(s) including viral inactivation procedures, which reduces the risk for contamination of the recombinant protein with human pathogens. Since human recombinant proteins produced in human cell lines carry a human glycosylation pattern, they are also less susceptible to degradation in comparison to human proteins lacking their natural glycosylation pattern. In summary the method of the invention offers various advantages over the prior art.

In particular, the method of embodiment (7) of the invention provides an effective system to produce safe and highly active human recombinant blood clotting factors, for example factors IX and FVIII for therapeutic application for Hemophilia B and A in humans. The method is suitable for expression of those wild-type proteins, but can also be used for mutants of those proteins, for example of factor VIII, which are exceptionally stable against proteolytic inactivation and thus allow to be subjected to vigorous virus inactivation protocols.

A preferred mode the method of embodiment (7) of the invention comprises serum-free culturing an immortalized cell line carrying a vector having a promoter linked to the 5′ end of a DNA sequence encoding said human blood protein. The 3′ end of the DNA sequence encoding said human blood protein is functionally linked to a bovine growth hormone polyA signal. According to the invention the immortalized human cell line is stably transfected with the vector. To detect stable transfection, the vector may further comprise, in addition to the gene for the human blood protein, at least one gene for a selection marker system which is functionally linked to a promoter.

Suitable promoters include viral promoters, housekeeping gene promoters, tissue specific promoters, etc. In case the promoter is a viral promoter, the cell line does not comprise the matching viral transcription activator protein for said promoter. However, the cell may comprise a viral transcription activator protein such as the T antigen which complements another viral promoter which is not functionally linked to the gene encoding the human blood protein. Preferably the promoter is a SV40 promoter, CMV promoter, EF-1alpha promoter, HSV TK promoter etc., most preferably the promoter is a CMV promoter, i.e. the constitutive, major intermediate early promoter of cytomegalovirus.

The expressions “transfection” or “transfected” refers to the introduction of a nucleic acid into a cell under conditions allowing expression of the protein. In general the nucleic acid is a DNA sequence, in particular a vector or a plasmid carrying a gene of interest under a suitable promoter, whose expression is controlled by said promoter. However, the term transfection also comprises RNA transfection. The skilled artisan is familiar with the various transfection methods such those using carrier molecules like cationic lipids such as DOTAP (Roche), DOSPER (Roche), Fugene (Roche), Transfectam® (Promega), TransFast™ (Promega) and Tfx™ (Promega), Lipofectamine (Invitrogene) and 293Fectin™ (Invitrogene), or calcium phosphate and DEAE dextran. He is also familiar with brute-force transfection techniques. These include electroporation, bombardment with nucleic-acid-coated carrier particles (gene gun), and microinjection. Finally the skilled artisan is also familiar with nucleic acid transfection using viral vectors.

“Transiently transfected” or “transient transfection” refer to the transient, i.e. non-permanent expression of the gene of interest due to the episomal nature of the introduced nucleic acid. By its very nature, RNA transfection or cytolytic viruses can only be used for transient expression. Episomal nucleic acids, including DNA (plasmids or vectors), is degraded by the cells after two to four days, and hence the expression of the gene of interest ceases then.

“Stably transfected” or “stable transfection” refers to the permanent expression of the gene of interest due to the integration of the transfected DNA into the genome of the cell. Most if not all cells have the potential to incorporate episomal DNA into their genome albeit at a very low rate. However, sophisticated selection strategies are employed to expand those cells that have integrated the transfected DNA. For that the vector must contain at least one gene for a selection marker such as e.g. hygromycin.

The term “stable transfection” or “stably transfected” is here also used to refer to cells carrying plasmids that can autonomously replicate and thus can be used for long-term expression of foreign genes. One particularly gene transfer system applicable for “stably transfecting” cells is based on recombinant retroviruses. Since integration of the proviral DNA is an obligatory step during the retroviral replication cycle, infection of cells with a recombinant retrovirus will give rise to a very high proportion of cells that have integrated the gene of interest and are thus stably transfected.

The term “culturing” refers to the maintenance of cells/cell lines in vitro in containers with medium supporting their proliferation and gene expression. Thus the culturing causes accumulation of the expressed secretable proteins in the culture medium. The medium normally contains supplements stabilizing the pH, as well as amino acids, lipids, trace elements, vitamins and other growth enhancing components.

The “serum-free”, “serum-free transfection” or “serum-free cultivation” refers to the transfection and culturing of cells in medium containing suitable supplements except any kind of serum. Supplements are selected from amino acids, lipids, trace elements, vitamins and other growth enhancing components. Often the “serum-free” culture conditions are even more stringent and, if no exogeneous protein is added, or already included in the medium, the medium is called “protein-free”.

The term “immortalized human cell line” refers to human cells that are not primary cells taken directly from an organism. In particular it refers to permanently established cell culture that will proliferate indefinitely given appropriate fresh medium and space, and thus have escaped the Hayflick limit.

The term “concentration” refers to the concentration of the produced recombinant protein from the culture medium. Inherently it also results in a concentration of the protein. The person skilled in the art is familiar with concentration techniques such as filtration, including ultra filtration, centrifugation, precipitation, etc. The concentration does not necessarily result in a pure protein, and the isolated protein may still comprise non-protein and protein contaminants. Additional purification steps are often required.

The term “purification” refers to steps applied to the isolated protein is subjected to in order to obtain a substantially pure (at least 60% pure, preferably at least 75% pure, more preferably over 90% pure and most preferably over 99.9% pure) human recombinant protein. Purity can be measured by an appropriate method. The person skilled in the art is familiar with techniques employable for the purification of a recombinant protein such as immuno-affinity chromatography, affinity chromatography, protein precipitation, buffer exchanges, ionic exchange chromatography, hydrophobic interaction chromatography, size-exclusion chromatography, electrophoresis. In addition, the purification may comprise a virus inactivation step such as heat treatment and/or solvent detergent (SD)-treatment, at either dry or liquid state, in the presence or without chemical substances including protease inhibitors. Further, the purification may include one or more steps for prion removal, such as protein precipitation, filtration, chromatography steps, in particular affinity chromatography steps (see e.g. “Partitioning of TSE infectivity during ethanol fractionation of human plasma”, Gregori, L. et al., Biologicals 32 1-10; 2 (2004); and “Removal of TSE agents from blood products”, Foster, P. R., Vax Sanguinis 87 (Suppl. 2), S7-S10 (2004)). After virus inactivation a further purification step selected from anyone of the above listed ones may be necessary for removal of the chemical substances used for virus inactivation.

The term “vector” refers to any genetic construct, such as a plasmid, phage, cosmid, etc., which is capable of replication when associated with the proper control elements, into which fragments of DNA may be inserted or cloned. A vector comprises unique restriction sites and may be capable of autonomous replication in a host cell. The term includes cloning and expression vehicles. The “vector” may further carry one or more further regulatory elements, said regulatory elements preferably being selected from splice sites, recombination sites, polyA sites, enhancers, multicloning site and prokaryotic plasmid sequences.

The term “functionally linked” refers to the configuration of the vector where the promoter is located within the vector in such a manner that it can stimulate transcription of the DNA sequence coding for the protein of interest, in particular for the human blood protein.

The term “mature” refers to the molecular structure of a given protein of the processed protein, i.e. a protein which lacks the N-terminal export signal.

The term “promoter” refers to a region of a regulatory DNA sequence bound by RNA polymerase and transcription factors during the initiation of transcription.

The term “enhancer” refers to a cis-acting sequence that increases the utilization of an eukaryotic promoter, and can function in either orientation and in any location (upstream or downstream) relative to the promoter.

The term “polyadenylation (polyA) signal” refers to a specialized termination sequence. It signals the addition of a “tail” of adenines to the end of the mRNA that enables export of the mRNA to the cytoplasm. Upon reaching the cytoplasm, the polyA tail of the mRNA is maintained during protein translation and stabilizes the mRNA during protein expression.

The term “encodes” or “encoding” refers to a property of the nucleic acid sequence to be transcribed (in case of DNA) or translated (in case of mRNA) into a polypeptide (protein) in vitro or in vivo when placed under the control of an appropriate regulatory sequence.

For the purpose of the present application the term “express”, “expressing” or “expression” refers to the transcription and translation of a gene encoding a protein.

The “human proteins” of the invention include, but are not limited to human proteins, polypeptides, mutations and modifications thereof. In particular the human proteins include recombinant plasma proteins, e.g. blood clotting factors (such as factor VIII, Factor VII/VIIa, Factor V, factor IX, Factor XI, von Willebrand factor, etc.), growth factors (such as erythropoietin, etc.), colony-stimulating factors (CSFs) (such as granulocyte stimulating factor (G-CSF), macrophage CSF (M-CSF), granulocyte-macrophage CSF (GM-CSF), cytokines (such as interleukins including interleukin 3, etc.), protease inhibitors (such as alpha-1-antitrypsin (A1AT), chymotrypsin, etc.), transport proteins (such as hormones, etc.), inhibitory or regulatory acting proteins, and the like. Furthermore mutations and modifications of these proteins or polypeptides are included, specifically mutations or modifications providing for a better stability of the recombinant protein, an elongated half-life, or a better recovery and include deletion, substitution or insertion mutants and chemical mutations of functional groups, respectively. Particularly preferred proteins which can be produced by the method of the invention of the application are human factor VIII (including B-domain deleted or wild-type), human factor IX, human G-CSF, human A1AT, human factor VII/VIIa and von Willebrand factor.

The recombinant production of the factor VIII and IX is known in the art (EP-A-160457; WO-A-86/01961, U.S. Pat. Nos. 4,770,999, 5,521,070 and 5,521,070).

In the case of factor VIII recombinant expression of subunits for the production of complexes showing coagulant activity is known in the art (e.g., from EP-A-150735, EP-A-232112, EP-A-0500734, WO-91/07490, WO-95/13300 U.S. Pat. Nos. 5,045,455 and 5,789,203). Moreover, the expression of truncated cDNA-versions partially or entirely lacking the sequence coding for the highly glycosylated B-domain have been described (e.g. in WO-86/06101, WO-87/04187, WO-87/07144, WO-88/00381, EP-A-251843, EP-A-253455, EP-A-254076, U.S. Pat. Nos. 4,868,112 and 4,980,456, EP-A-294910, EP-A-265778, EP-A-303540 and WO-91/09122). A particular factor VIII mutant in which the B-domain between positions Arg740 and Glu1649 has been replaced by an Arg-rich linker peptide having at least 3 Arg residues and comprising 10 to 25 amino acid residues (wherein said factor VIII numbering is relative to the mature wild-type factor VIII sequence shown in SEQ ID NO:9) is disclosed in WO 01/70968 which is herewith incorporated in its entirety. In particular, the Arg-rich linker peptide has 14 to 20 amino acid residues, while a linker comprising:

-   -   the amino acid sequence SFSQNSRH (SEQ ID NO:10), and/or     -   the amino acid sequence QAYRYRRG (SEQ ID NO:11), and/or     -   the amino acid sequence SFSQNSRHQAYRYRRG (SEQ ID NO:12)         is particularly preferred. Such B-domain factor VIII mutein is         encoded by nt 783 to 5162 of SEQ ID NO:3.

G-CSF is a lineage specific, small molecule in human blood that stimulates the production of a type of white blood cell from the bone marrow, known as neutrophils. Neutrophils play a central role in the body's immune system and defend infections. G-CSF (particular cDNA sequences of the a, b and c form thereof being given in SEQ ID NOs:15, 16 and 17, respectively; the protein of the G-CSF b form (hereinafter “G-CSFb” protein) is shown in SEQ ID NO:27) is naturally produced by monocytes, fibroblasts, and endothelial cells. Normally the concentration in blood is about 40 pg/ml in healthy persons. In patient plasma, the level of G-CSF can drop more than ten-fold. G-CSF is also produced in cancer cell lines like 5637 cells which secrete about 70 ng/ml. For therapy, recombinant human G-CSF is produced in E. coli as a N-terminal methylated, non-glycosylated form by Amgen Inc. (Filgrastim/Neupogen®), which is also available as a PEGylated product (Pegfilgrastim/Neulasta®). Another drug is produced in CHO cells by Chugai Pharmaceuticals Co, which results in a glycosylated product (Lenograstim/Granocyte®). G-CSF is used as a drug to treat neutropenia either inherited or caused by chemotherapy (cancer), AIDS or bone marrow transplantation. For this, a typical dose is 5 μg/kg and day.

A particular A1AT cDNA sequence suitable with the invention of the present application is given in bps 973 to 2259 of SEQ ID NO:2. Particular factor VII/VIIa cDNA sequences are given in SEQ ID NOs:13 and 14 corresponding to the a and b form thereof. A particular vWF cDNA is given in SEQ ID NO:18.

The selection marker system includes hygromycin resistance, puromycin resistance, neomycin resistance, adenosine deaminase (ADA) resistance, aminoglycoside phosphotransferase (neo, G418, APH) resistance, bleomycin (phleo, bleo, zeocin) resistance, cytosine deaminase (CDA, CD) resistance, cytosine deaminase (CDA, CD) resistance, dihydrofolate reductase (DHFR) resistance, histidinol dehydrogenase (hisD) resistance, hygromycin-B-phosphotransferase (HPH) resistance, puromycin-N-acetyl transferase (PAC, puro) resistance, thymidine kinase (TK) resistance, and Xanthine-guanine phosphoribosyltransferase (XGPRT, gpt) resistance. Particularly preferred is the hygromycin resistance gene. Also the gene for the selection marker may be functionally linked with a polyA signal such as the one derived from the bovine growth hormone (BGH) or the SV40 polyadenylation signal.

The transfected cells are constantly exposed in their culture medium to the protein of the selection marker system, such as hygromycin during the selection phase, resulting in the survival of only those cells carrying the vector. A person skilled in the art is familiar with alternative selection markers suitable for the establishment of stably transfected cells, as well with the concentrations of the chosen selective agents which needs to be applied.

A particularly preferred vector of the invention carries a CMV promoter, a hygromycin gene, a polyA sequence and the gene of interest and preferably is the pcDNA3.1 vector of Invitrogen having the sequence of SEQ ID NO:4 wherein resequencing said vector it was found that it in fact has the sequence shown in SEQ ID NO:5.

The immortalized cell lines suitable for the method of the invention are selected from the group of kidney, bladder, liver, lung, cardiac muscle, smooth muscle, ovary or gastrointestinal cells. Those cells may carry in their genome adenoviral DNA sequences, in particular the first 4344 nucleotides of Ad5 sequences. Preferred are human foetal kidney cells (HEK) selected from the group consisting of 293 cells (ATCC CRL-1573; DSM ACC 305; ECACC ref.: 85120602), 293T cells (DSM ACC 2494; ECACC: tsa201, ref. 96121229), and FreeStyle 293 cells (293F cells; Invitrogen R79007). Most preferred are 293F cells. Those immortalized cell lines carrying said vector are cultured under conditions allowing expression of the recombinant gene. Essentially those are standard culturing conditions known to the person skilled in the art, however in case of cells carrying the gene for human factor IX, vitamin K should be included in the medium.

A particular embodiment of the present invention is the serum-free production of the recombinant protein in serum-free culture of the stably immortalized cells, which are also transfected under serum-free conditions. For that anyone of the above described immortalized human cell lines, preferably the 293F cell line is transfected and cultured under serum-free conditions. The cells are stably transfected in suspension culture in the absence of serum and then adapted to adherent cell growth for selection of single cell clones. Once individual clones are obtained, they are expanded adherently. After selection of best producing clones the cells are transferred to suspension culture. During the whole stable cell line procedure and in further up-scaling for production, cells are grown in serum-free medium and are never in touch with serum or human or animal proteins. The recombinant blood protein such as anyone of the blood clotting factors or a protease inhibitor such as A1AT or growth factors (such as G-CSF and GM-CSF) are isolated from the culture broth, and standard purification steps do follow. In more detail, the particular embodiment of serum-free production of the recombinant human blood protein, in particular human factor VIII or factor IX or A1AT or G-CSFb comprises the following steps:

(1) Transfection of human immortalized cells, preferably 293F cells in suspension culture without serum. Cells are cultured in disposable, sterile polycarbonate Erlenmeyer flasks. Cells are transfected with a density of e.g. 1×10⁶ viable cells per ml with a transfection agent, preferably a cationic transfection agent, more preferred lipofectamine 2000 CD reagent (Invitrogen) or a reagent for the calcium phosphate transfection method); a vector is transfected encoding the human blood protein, preferably the vector is pcDNA3.1-FIX, pcDNA3.1-FVIII, pcDNA3.1-A1AT or pcDNA3.1-GCSFb; (2) 24 to 120 h, preferably 36-96, more preferably 48 h post-transfection a suitable number of cells (10³ to 10¹⁰; preferably 10⁵ to 10⁸, most preferably 10⁶ cells) are transferred into a flat culture dish for sedimentation to establish adherent growth. Preferably the culture dish is a 10 cm-dish and cells are cultured in serum- and protein-free media, preferably FreeStyle 293 Expression medium (12338-018, Invitrogen) or the serum-free in-house medium (Octapharma Stockholm). (3) Selection pressure is started at 2 to 50 h, preferably 48 h post transfer into the flat culture dish. The medium is supplemented with a suitable selective agent selected from the group consisting of selection markers, e.g. hygromycin, neomycin, G418 and Zeocin. The preferred selective agent is hygromicin with a concentration of 10 to 300 μg/ml, preferably 50 to 200 μg/ml, most preferably 50 μg/ml. The pressure is maintained for at least 10 to 20 days, preferably for 14 days, whereby the hygromycin supplemented medium is exchanged every other day. Only stably transfected cells survive these selection conditions and form adherent cell clones which can be individually picked. Additionally an attachment factor can be used in order to stick cells on the dish and prevent cells floating from one clone to an other cell clone. This attachment factors could be f. e. poly-D-Lysine, synthetic medium supplements without human or animal proteins, or other substances. Alternatively, cloning rings could be used for picking of clones. (4) Individual cell clones are picked and transferred into separate culture containers for serum-free expansion of cells (scaling up) while the selective pressure is omitted. Any culture container is suitably, but preferably the individual clones are first transferred into 96 well plates with a sufficient amount of medium, and then to 48-, to 24-, to 12- and to 6-well plates and then to spintubes. At the spintube stage, the cells are cultured in serum-free medium softly shaking in order to bring cells back into suspension growth. Once the cells have reached the 6-cell well plate or the spintube stage, it is optional to select the best cell clones according to some selection criteria, i.e. the growth rate of the cells, faster growing cells are preferred, and the amount of the recombinant protein they do produce. However, said selection can also be performed at any later stage. (5) Cells obtained from the spintube culture were seeded into Erlenmeyer culture vessels with a sufficient amount of serum free medium. Additional selection criteria for up-scaled, in suspension growing serum- and protein-free cell clones are as follows: viability, cell morphology, no aggregation, robustness concerning centrifugation and no cell debris.

The method of the present invention works particularly well if the vector is pcDNA3.1-hygro(+)-zz. It is preferred that the gene encoding the human protein, in particular human FIX, FVIII, A1AT or G-CSFb is inserted in such a way that it is under the control of the CMV promoter as it is shown in FIGS. 2, 3, 7, and 11 respectively. Preferably the wild-type sequences of said genes are inserted, such that the recombinantly expressed protein is without any mutation and is structurally identical to the wild-type protein isolated from blood plasma. A schematic drawing of the wild-type human factor IX is shown in FIG. 1. SEQ ID NOs:1, 2, 3 and 22 provide the nucleic acid sequence for pcDNA3.1-FIX, pcDNA3.1-A1AT, pcDNA3.1-FVIII and pcDNA3.1-GCSFb, respectively. The respective proteins are encoded by nucleotides 939 to 2224, 913 to 2259, 679 to 5055 and 970 to 1584, respectively.

The present invention thus provides a method for the recombinant production of human factor IX, A1AT, factor VIII and G-CSFb cloned into pcDNA3.1™ giving rise to pcDNA3.1-FIX, pcDNA3.1-A1AT, pcDNA3.1-FVIII and pcDNA3.1-GCSFb, respectively, which are integrated into the genome of immortalized human cells, preferably human embryonic kidney cells such as 293 cells (ATCC CRL-1573; DSM ACC 305; ECACC ref.: 85120602), FreeStyle 293 cells (293F cells; Invitrogen R79007) or 293T cells (DSM ACC 2494; ECACC: tsa201, ref. 96121229).

Those cells carrying either pcDNA3.1-FIX or pcDNA3.1-A1AT or pcDNA 3.1-FVIII or pcDNA 3.1-GCSFb are cultured in medium under standard conditions enabling gene expression or alternatively they are cultured under serum-free conditions to minimize the risk of contamination with human pathogens. One or more prion removal steps may be included, such as protein precipitation, filtration, chromatography steps, in particular affinity chromatography steps. Alternatively/additionally a prion knock-out cell line can be used as expression cell. This can be obtained by complete genomic knock-out or antisense technology. In case of the production for human factor IX the cells are preferably cultured in the presence vitamin K. The human blood protein is isolated from the culture supernatant and subjected to subsequent purification steps known in the art to maximize the yield of a pure, stable and highly active product and are selected from immunoaffinity chromatography, anion exchange chromatography, size exclusion chromatography, etc., and combinations thereof. They can easily be adapted to the specific requirements needed to isolate recombinant factor IX, G-CSFb or A1AT. Quantity and activity of the purified protein during and after the purification procedure may be monitored by ELISA and/or one-stage coagulation time assays (aPTT).

To overcome the problems of possible infectious contaminations in the purified protein samples or in the product directly obtained from the cell culture supernatant containing the secreted recombinant protein of choice, the culture supernatant might be treated with procedures for virus inactivation including heat treatment and/or SD-treatment (dry or in liquid state, with or without the addition of chemical substances including protease inhibitors). A person skilled in the art is familiar with purification procedures. For example, the isolation and purification and recovery of high purity virus-inactivated factor VIII from blood plasma by anion exchange chromatography was described I (WO93/15105). In addition several processes for the production of high-purity, non-infectious coagulation factors from blood plasma or other biological sources have been reported. Lipid coated viruses are effectively inactivated by treating the potentially infectious material with a hydrophobic phase forming a two-phase system from which the water insoluble part is subsequently removed. A further advantage has been proven to complement the hydrophobic phase treatment simultaneously or sequentially with a treatment with a non-ionic biocompatible detergents and dialkyl or trialkyl phosphates (WO 96/36369, EP 0131740, U.S. Pat. No. 6,007,979). Non-lipid coated viruses require inactivation protocols consisting in treatment with non-ionic detergents followed by a heating step (60-65° C.) for several hours (WO 94/17834). After virus inactivation, a further purifying step for removing the chemical substances may be necessary. In summary, the present invention provides an effective protein production method based on a human cell line linked to approved methods of protein purification and for inactivation of potentially dangerous infectious agents. A safe and easy to use-system for production of recombinant proteins, for example the blood clotting factor IX or VIII, A1AT and G-CSFb has been established. The activity of the recombinantly produced proteins can be examined with standard tests. In case of the human factor IX for example with an activated partial thromboplastin time assay using Dapptin TC (Kaolin/Sulfatid-Phospholipid Cat. No. 5035090, Technoclone GmbH) activation with a manual coagulation instrument. Finally the thus obtained recombinant proteins, such as the blood protein described hereinbefore, in particular the human factor IX may be used in a pharmaceutical composition.

The invention is further described in the following examples. Said examples are however not to be construed as to limit the invention.

EXAMPLES Materials and Methods

Human Cell Lines for Protein Expression:

Preferred cell lines are HEK293 (ECACC Ref. 85120602), FreeStyle 293 (293F; Invitrogen R79007) and 293T (tsA201, ECACC Ref. 96121229) which is a transformed embryonic human kidney cell line stably expressing an SV40 temperature-sensitive T antigen. These epithelial-like cell lines have been used in a variety of functional expression assays and been reported to produce high levels of recombinant proteins. The 293F cell line (Invitrogen), which is derived from the 293 cell line was preferably used in the Examples below. The parental cell line 293 is a permanent line established from primary embryonal human kidney transformed with sheared human adenovirus type 5 DNA (Graham et al., 1977; Harrison et al., 1977). The 293F cell line is a variant of the 293 cell line that has been adapted to suspension growth in FreeStyle™ 293 (293F) Expression Medium (12338-018, Invitrogen). The 293F cell line was obtained from Robert Horlick at Pharmacopeia. The 293F cell line was originally prepared from low passage Master Cell Bank cultures derived from the parental 293F cells that were re-cloned by limiting dilution. Cells have been constantly grown in the serum-free FreeStyle 293 Expression medium or a serum-free medium (Octapharma Stockholm) with good viability and good morphology for more than one year during the development of the present invention.

For efficient production of human factor IX, the medium can be modified by addition of vitamin K. These cell lines are capable of being cultivated in serum-free and/or protein-free medium containing suitable supplements.

Determination and Measurement of Target Proteins

Determination of Human Factor IX Concentration by ELISA:

Human recombinant factor IX levels in the supernatant were determined by ELISA using a goat anti-human FIX (GAFIX-AP, Affinity Biologicals) as capture antibody according to standard procedure. All incubations were performed in a humid chamber at RT. Both standards, Octanyne (plasma-derived FIX, Octapharma) and BeneFIX (recombinant FIX, Genetics Institute) were used. The detecting antibody was a peroxidase conjugated goat anti-human FIX (GAFIX-APHRP, Affinity Biologicals). ABTS (Cat. No. 1682008, Roche Diagnostics) was added to each well as substrate, colorimetric reaction was detected at 405 nm in 15 minutes. Results were calculated by linear regression of standard concentration versus standard absorbance.

Detection of Human Clotting Factor IX Activity:

The clotting activity of human recombinant factor IX in supernatants was determined as follows: The clotting activity was assayed based on an activated partial thromboplastin time assay using Dapptin TC (Kaolin/Sulfatid-Phospholipid, Cat. No. 5035090, Technoclone GmbH) activation with a manual coagulation instrument (Amelung KC 4A micro, Amelung GmbH). For the study, 50 μl supernatant from transfected cells, 50 μl FIX-deficient plasma (Progen) and 50 μl Dapptin TC were incubated for 2 minutes at 37° C. Coagulation was started by adding 50 μl CaCl₂ (Cat. No. 84687-22F, Instumentation Laboratory). Sample coagulation time was compared to both Octanyne or/and BeneFIX.

Determination of BDDrhFVIII with COAMATIC: FVIII Assay (Chromogenix):

The commercial chromogenic assay kit COAMATIC:FVIII (Chromogenix, cat. No. 82 25 85) contains FIX, FX and a chromogen which is turned into a yellow water soluble dye by FXa cleavage. FVIII containing samples complete this system: FVIII activates FIX by complexing, this complex activates FX by proteolytic cleavage to become FXa. FXa turns the chromogen into a dye which subsequently is determined photometrically at 405 nm. This test is designed for determination of FVIII from patient plasmas. The following procedure was set up in order to make this test applicable for the factor VIII measurement in diluted culture media. As control standards, full length recombinant human clotting factor VIII (NIBSC, order no. 57814F) and normal control plasma (Instrumentation Laboratory Company) was used.

Sample Preparation:

Samples were diluted with dilution buffer delivered with COAMATIC reagents to a prospective final FVIII activity between 2 and 20 mIU/ml and are compared to the WHO No. 6 standard curve.

Method:

On a 96-well array placed on the thermobloc, both standards and samples are measured in triple.

Operation scheme (per well): Reagent Volume Incubation (37° C.) Diluted standards and 50 μl 4 min samples Factor reagent (37° C.) 50 μl place into 96-well photometric device, incubate 2 min at 37° C. in incubator S-2765 + I-2581 50 μl place into 96-well photometric device, (37° C.) incubate 2 min at 37° C. in incubator 20% acetic acid 50 μl place into 96-well photometric device, shake 15 s in 96-well photometer determine absorptions at 405 nm immediately

Determination of A1AT Activity with Elastase Activity Test:

After transfection of A1AT cDNA, A1AT was expressed and secreted into cell culture medium. After removal of cells by centrifugation (5 minutes, 1000 rpm), A1AT activity was measured in culture supernatant. In this activity test, A1AT activity was determined by its inhibitory effect upon elastase. Elastase cleaves pNA from the substrate N-succinyl-(Ala)₃-pNA. pNA release is measured photometrically at 405 nm. By comparison with standard samples with defined A1AT activity, the activity of the respective samples is determined. As proven in other experiments, the test is valid in serum-free Freestyle medium. To confirm the fact that Freestyle medium has no influence on the test, two standard curves were prepared: standard human plasma was diluted in T+buffer or in Freestyle medium.

Dilution of Samples:

All samples were tested undiluted, 1:10 and 1:50 diluted in Freestyle medium and are compared with standard dilutions of human plasma.

Method:

50 μl of each standard dilution and sample dilution, respectively was pipetted into a well of the 96-well micro titer plate. After adding 150 μl of Elastase working solution to each well, the 96-well plate was shaken for 1 minute on the ELISA reader and incubated for 30 minutes at 37° C. 100 μl of substrate working solution was added to each well with the multipette. Absorption at 405 nm was measured immediately after addition of substrate solution and after 7 minutes incubation at 37° C. in the dark. The first value represents the basis absorption without elastase-catalised reaction and is substacted from the second one which represents the absorption after elastase cleaved pNA from the substrate. Using the result after the substraction, the A1AT activities of the samples are calculated according to the standard curve.

Determination of G-CSF Activity by ELISA:

Human recombinant G-CSF levels in the cell culture supernatants were determined by ELISA using a mouse anti-human G-CSF antibody (MAB-214, R&D System) as capture antibody according to standard procedure. All incubations were performed in a humid chamber at room temperature. The G-CSF standard (recombinant hG-CSF, E. coli, 214-CS-025, R&D Systems) was used. The detection antibody was a biotinylated goat anti-human G-CSF (BAF-214, R&D Systems). Streptavidin was conjugated to horseradish-peroxidase (DY998, R&D Systems) linked to the detection antibody. The QuantaBlu™ Fluorogenic Peroxidase Substrate (15169, Pierce) was added to each well as substrate, fluorometic reaction was detected at extinction 320 nm/Emission 420 nm within 60 min. Results were calculated by linear regression of standard concentration versus standard relative fluorescence units (RFU).

Example 1 Cloning of Target Proteins

A. Cloning of Human Factor IX:

From the vector pTG36 as disclosed in WO01/70968, a 1.4 kb fragment containing the open reading frame of the human clotting factor IX was cut out by double-digestion with Hind III and NotI. This fragment was ligated to the 5.6 kb fragment of the HindIII and NotI double-digested vector pcDNA3.1Hygro(+)-zz (derived from V870-20, Invitrogen) resulting in the vector pcDNA3.1-FIX shown in FIG. 2. The DNA sequence of pcDNA3.1-FIX is shown in SEQ ID NO:1. Three additional nucleotide insertions in the vector backbone were found in the pcDNA3.1Hygro(+)-zz vector (see SEQ ID NO:5) compared to the sequence published by Invitrogen (as shown in SEQ ID NO:4). The vector pcDNA3.1-FIX contains a cassette hygromycin-resistance gene to enable a selection method for a stably transfected cell clone (see FIGS. 2, 3 and 7). The vector allows the establishment of stably expressing cell lines by calcium phosphate transfection or others, and subsequent selection for hygromycine resistants.

B. Cloning of Human Factor VIII:

A 4380 bp FVIIIcDNA containing the open reading frame of a B-domain deleted human clotting factor VIII was isolated from the vector pTGF8-2hyg-s (SEQ ID NO:7; the production of which being disclosed in WO01/70968) with NotI+XhoI digestion and ligated with pcDNA3.1Hygro(+)-zz, which was linearized with XhoI+PspOMI resulting in the vector pcDNA3.1-FVIII shown in FIG. 7.

C. Cloning of Human A1AT:

A1AT mRNA was isolated directly from the HepG-2 cells (DSMZ#ACC 180) using mRNA Miniprep Kit (Sigma, Cat#MRN-10). In the following step mRNA was captured on oligo (dT) beads. Afterwards, mRNA will be transcribed into double-stranded cDNA with Avian Myeloblastosis Virus Reverse Transcriptase (AMV RT, Promega, Cat#M5101) following RT-PCR (reverse Transcription-Polymerase Chain Reaction). A1AT cDNA was amplified with PCR reaction. The PCR product was loaded on agarose gel. The appropriate DNA-band was isolated and afterwards purified with the Qiaquik Gel Extraction Kit (Qiagen, Cat#28704). Then A1AT fragment was subcloned into a commercial Vector (TOPO® Invitrogen, Cat#K4650-01). For cloning of pCMV-Script: PCR II TOPO-A1AT was digested with EcoRI, the A1AT 1370 bp fragment was ligated with pCMV-Script linearized with EcoRI.

For cloning of pCI-neo-A1AT PCR II TOPO-A1AT was digested with EcoRI, the A1AT 1370 bp fragment was ligated with pCI-neo linearized with EcoRI.

For cloning of pcDNA3.1-FVIII 1370 bp A1AT was isolated with PCR II TOPO-A1AT digested with XhoI+HindIII and ligated with pcDNA3.1 linearized by XhoI+HindIII. The resulting vector is shown in FIG. 3.

For cloning of pTG1-A1AT PCR II TOPO-A1AT was digested with HindIII and NotI. The A1AT 1370 bp fragment was ligated with pTG1 (no PRE), linearized with HindIII and NotI. The resulting vector is shown in FIG. 9.

D. Cloning of Human G-CSF cDNA:

Total RNA was isolated directly from natural 5637 human urinary bladder carcinoma cells with RNeasy mini kit (QIAGEN, cat. No. 74104). Afterwards, the isolated total RNA was incubated with DNase I to digested possibly mixed genomic DNA of 5637 cells. To get DNase-free total RNA the reaction mixture was treated with RNeasy clean-up kit (QIAGEN, cat. No. 74204). RT-PCR with the total RNA as template was performed with oligo(dT)12-18 primer (Invitrogen, Cat. No. 18418-012) and Superscript™ II RNase H—Reverse Transcriptase (Invitrogen, Cat. No. 18064-022) in the presence of RNase inhibitor (Roche, Cat. No. 799-017) to synthesize ds cDNA pool from 5637 cells. G-CSF cDNA was amplified then with PCR reaction. G-CSF cDNA was isolated from agarose gel with QIA quick Gel Extraction kit (QIAGEN, Cat. No. 28704) and sequenced with both of the following G-CSF PCR primers:

-   -   G-CSF Forward: 5′-ATG GCT GGA CCT GCC ACC CAG AGC-3′ (SEQ ID         NO:19)     -   G-CSF Reverse: 5′-TCA GGG CTG GGC AAG GTG GCG TAG-3′ (SEQ ID         NO:20)

The sequence of the DNA synthesized from 5637 cells was confirmed by sequence analysis to be a GCSF-b form (having the sequence shown in SEQ ID NO:26).

The cDNA of GCSF-b form isolated as described above was then directly ligated into the commercial vector pCR2.1 (Invitrogen). The resulting plasmid was designated pCR2.1d2-GCSFb and is shown in FIG. 10.

PCR2.1d2-GCSFb was digested with HindIII and NotI, the 705 bp GCSFb cDNA fragment was isolated and ligated into the vector pcDNA3.1Hygro(+)-zz, which was linearized with HindIII and NotI. The resulting pDNA3.1-GCSFb vector is shown in FIG. 11.

PCR2.1d2-GCSFb was digested with EcoRI, the 629 bp GCSFb cDNA fragment was isolated and ligated into the pCINeo vector, which was linearized with EcoRI. The resulting pCINeo-GCSFb vector is shown in FIG. 12.

PCR2.1d2-GCSFb was digested with BamHI and XhoI, the 693 bp GCSFb cDNA fragment was isolated and ligated into the pCMVScript vector, which was linearized with BamHI and XhoI. The resulting pCMVScript-GCSFb vector is shown in FIG. 13. PCR2.1d2-GCSFb was digested with HindIII and NotI, the 705 bp GCSFb cDNA fragment was isolated and ligated into pTG2-hyg-as vector, which was linearized with HindIII and NotI. The resulting pTG2-GCSFb-hyg-as vector is shown in FIG. 14.

Example 2 Expression of Target Proteins in Different Cell Lines and Different Vectors

When optimizing the present method for recombinant protein production the ability for high levels of expression of different cell lines—all carrying a vector comprising the recombinant gene for Alpha-1-antitrypsin (A1AT)—was tested. CHO, BHK and other cell lines were found to produce less recombinant protein in transient transfection assays compared to the 293T cell line. Therefore other human embryonic kidney cell line derivates were examined. The results are shown in FIGS. 4 to 6.

Example 3 Transient Transfection of 293T and 293 Cells in Serum Containing Medium as Comparison to 293F Cells, which were Transfected and Cultured Under Serum-Free Conditions

0.1-0.2×10⁶ viable cells of 293T or 293 cells were plated into 6-well. On the next day cells were transfected using Calcium phosphate method (Biotechniques 6:7 632-638 (1988)): 4 μg of plasmid DNA were diluted in 0.1×TE buffer (ad 200 μl transfection mix), mixed gently, 20 μl 2.5 M CaCl₂ and 100 μl 2×HBS were added to the transfection sample. The transfection sample was incubated for 20 min at room temperature. After 6 h incubation medium was exchanged and cells were then incubated for 48 h.

Example 4 Serum-Free Transfection and Expression of Target Proteins in 293F Cells in Serum-Free Medium

28 ml suspension culture was prepared with a cell density of 10⁶ viable 293F cells (on the same day of the transfection experiment). A lipid-DNA complex was prepared by diluting 30 μg of plasmid DNA in Opti-MEM® I (Invitrogen) to a total volume of 1 ml, and 40 μl of 293Fectin® was diluted in Opti-MEM® I to a total volume of 1 ml. After the 5 min incubation at room temperature, diluted DNA was added to 293Fectin® to obtain a total volume of 2 ml. The transfected samples have been incubated for 20 min at room temperature in the dark. 2 ml of the transfection mix was added to the 28 ml 293F suspension culture (final cell density is 1×10⁶ cells/ml). The transfected 293F cells were incubated at 37° C./humidified atmosphere of 8% CO₂ in air on an orbital shaker rotating at 125 rpm for 72 h.

A: Transfection and Expression of A1AT:

The results of those experiments comparing 293F with 293 and 293T cells are shown in FIG. 4. In all experiments a defined quantity of cells (10⁶ cells) were transfected with pcDNA3.1-A1AT. The amount of A1AT expressed in these different cell lines was compared. The expressed amount of A1AT in 293 F cells was set as 100%. As can be seen from FIG. 4, 293 and 293 T cells produced only 12-13% of the amount of A1AT than 293F cells.

Moreover, it was tested whether different vector backbones influence the amount of recombinant protein produced in 293F cells. The coding sequence for human Alpha-1-antitrypsin (A1AT) was inserted into pTG (in-house vectors), pCMV Script® (Stratagene), pcl neo (Promega) as well as into the pcDNA3.1™ vector.

The expression level of A1AT from pcDNA3.1-A1AT was set as 100%. None of the other vectors came close to the high expression observed with pcDNA™ 3.1-A1AT. It was found that pcDNA 3.1-A1AT produced the greatest amount of A1AT as detected with ELISA (see FIG. 5). The in-house vector expressed only an amount of 20% and the other commercial vectors revealed a range of 15-30% compared to the amount of A1AT expressed from pcDNA 3.1. Therefore pcDNA 3.1 was chosen for all further experiments.

In summary, different cell lines had been transiently transfected with pcDNA3.1™ carrying the A1AT gene. It was shown that the serum-free 293F cell line expresses 7-times more A1AT per 10⁶ cells than 293T and 293 cells. Therefore freestyle 293 F cell line were chosen for stable transfection experiments.

The results of transient transfection experiments are shown in FIG. 6. The top panel (A) shows the SDS-PAGE analysis of the supernatant of 6 different transfection trials using different transfection vectors. Derived from analytical Figures it can be concluded that the α1-antitrypsin present in the analysed cell culture supernatants is of good quality as can be deduced from the ratio of activity to antigen being 1 (data not shown). Validity of the test results can be deduced from the fact that the negative control does not show α1-antitrypsin activity nor antigen.

The molecular weight distribution analysed by SDS-PAGE shows well comparable pictures for the three α1-antitrypsin containing samples. In the negative control, besides the lack in the α1-antitrypsin representing band, an additional band at a molecular weight of 27 kD is visible as expected.

By analysis using western blotting (using an anti human α1-antitrypsin primary antibody) the protein can be identified in the expected molecular weight region under reducing conditions. Split products are not visible.

The black arrow points to the prominent band which corresponds to the 52 kDa recombinant protein alpha-1-antitrypsin. Also visible is a band corresponding to the 27 kDa GFP protein in lanes 4 and 8, which was transiently expressed as control in cell line freestyle 293 F cells. The additional bands in lane 1, 2, 3 and 5, 6 and 7 are host cell proteins (from freestyle 293 F cells). The lower panel (B) shows the Western Blot analysis. The results are identical except that due to the higher stringency of the assay, the results appear cleaner, and only the band corresponding to the A1AT is visible.

B: Transfection and Expression of FVIII:

In FIG. 8 the average amount of factor VIII of the best three stably transfected clones is shown. The average amount of factor VIII of the three best clones expressed with pcDNA-FVIII vector is set as 100% productivity. A comparison with the in-house vector pTGF8-2hyg-s reveals almost 3-fold higher productivity of factor VIII with pcDNA3.1-FVIII vector in 293F cells.

C: Transfection and Expression of FIX:

In stably transfected 293F cells using pcDNA3.1-FIX and a pUC 19/X based vector pTGF36 (see WO 01/70968) expressing factor IX, almost 3-fold higher productivity could be shown with the use of pcDNA3.1 vector in 293F cells as can be seen in the following Table 1.

Table 1:

FIX productivity in 293 and 293F cells after stable transfection with pcDNA3.1 and pTG2 vector

FIX productivity in % Substrate cell [mIU/10⁶ cells,day] line/vector pcDNA3.1-FIX pTGZ-FIX 293 (+serum) 100% — 293F (−serum) 100% 60%

Example 5 Production of G-CSFb

A. Transfection of 293F Cells in Serum-Free Medium, Transient Transfection:

28 ml suspension culture of 293F cells with a cell density of 1.1×10⁶ viable 293F cells per ml was prepared on the same day of the transfection experiment. A lipid-DNA complex was prepared by diluting 30 μg of plasmid DNA (pcDNA3.1-G-CSFb) in Opti-MEM® I (Invitrogen) to a total volume of 1 ml and 30 μl of Lipofectamine 2000 CD was diluted in Opti-MEM® I to a total volume of 1 ml. After 5 min incubation at room temperature, diluted DNA was added to Lipofectamine 2000 CD to obtain a total volume of 2 ml. The transfection samples were incubated for 20 min at room temperature in the dark. 2 ml of the transfection mix was added to the 28 ml 293F suspension culture (final cell density is 1×10⁶ cells per ml). The transfected 293F cells were incubated at 37° C./humidified atmosphere of 8% CO₂ in air on an orbital shaker rotating at 125 rpm for 72 h.

B. Stable Transfection:

72 h after transient transfection as set forth in A. above, a suitable number of cells (10⁵ and 10⁶ cells) were transferred into a flat dish for sedimentation to establish adherent growth. Selection pressure was started after 2 to 50 h, preferably 48 h post transfer into the flat dish. The preferred selective agent was hygromycine with a concentration of 75 μg/ml. The pressure was maintained for at least 10 to 20 days, preferably for 14 days, whereby the hygromcine supplemented medium was exchanged all 2 to 3 days.

C. Selection of Best G-CSF Producer Clones Using the Analysis and Picking Robot ClonePixFL (Genetix):

FreeStyle 293F cells stably transfected as described in B. above were seeded in semi-solid methyl-celluloses based medium containing an appropriate antibiotic for selection of clones after about two days and a labelled antibody for detection of the highest producer clones via fluorescence. Large numbers (thousands) of clones were analyzed using ClonePixFL (Genetix) with respect to the cell number and to G-CSF secretion in order to subsequently pick only a few hundred G-CSF best producer clones. In contrast to other known methods, where non-producer clones and mixed clones are randomly picked as well, the use of ClonePixFL allows picking of fast growing clones, which are high producers only, originated from single cells. The picked cells are expanded in microtiter plates and later in spin tubes, cell culture flasks and fermenters under serum-free conditions for the complete procedure.

Here as well the whole stable transfection procedure is generated under serum-free conditions. Additionally, during the whole following expansion and cell culture procedure, the cell did not have any contact to serum or animal derived proteins.

During expansion, the best clones are selected with respect to robustness, high growth rate, viability and production of active G-CSF as measured in ELISA format. After this selection phase, the picked clones are cultured under serum-free conditions without antibiotic supplements. 293F cells were cultured completely serum-free during the whole procedure, medium was exchanged every other day. Up-scaling of the cells was performed under completely serum-free conditions from Erlenmeyer flasks in Kühner Shakers to higher volumes in wave reactor (Wave Biotech Europe). During this selection the number is reduced again to only a few best producing clones. Correct cDNA sequence, mRNA content and behaviour upon fermentation are the criteria to identify the best clone(s) for subcloning. For this, cells of the selected clone(s) are plated, analyzed and picked with ClonePixFL, and then expanded and selected as described before. Subcloning is an essential step in order to select again for better producer clones to eliminate possible genetic variations in the plated subpopulation of the clone. After subcloning, the selected clone(s) are banked again under serum-free conditions. The expressed recombinant human G-CSF protein is characterized biochemically in more detail.

D. Determination of Human G-CSF Concentration by ELISA:

The quantity of the rhG-CSF expressed by the FreeStyle 293F cell lines thus obtained was determined by ELISA, and the yield of protein obtained with cells transfected with different vectors was compared (see FIG. 15). As expected from the expression experiments with FIX and A1AT described in Examples 2 and 3, here again a combination of the vector pcDNA 3.1-GCSFb with the 293F cell line showed the highest productivity and was therefore used for production of recombinant human G-CSFb.

E. Western Blot of rhG-CSF in Reducing SDS PAGE:

10 μl G-CSF produced in supernatants from HEK293 and HEK293F cells was analyzed on 15% SDS PAGE and western blot. Detection of G-CSF was done via BAF214-bio/SA-HRP/DAB (see FIG. 16). The arrow indicates the monomeric band of rhG-CSF of the correct molecular mass.

Sequence Listing, Free text Type Start End Name/Description SEQ ID NO: 1: pCDNA3.1-FIX, Molecule: 6960 bps DNA, circular REGION 209 863 CMV promoter REGION 895 911 MCS″ GENE 939 2324 hFIX GENE 2328 2339 SV40′/SV40 polya + intron REGION 2340 2370 ′MCS REGION 2381 2595 BGH pA REGION 2658 3071 f1 origin REGION 3136 3460 SV40 promoter GENE 3478 4501 HygR REGION 4514 4886 SV40 pA REGION 5819 5146 C PUC origin GENE 6824 5964 C AmpR(complementary strand) SEQ ID NO: 2: pCDNA3.1-A1AT Molecule: 6914 bps DNA, circular REGION 209 863 CMV promoter GENE 913 2259 A1AT GENE 2328 2339 SV40′/SV40 polya + intron REGION 2335 2549 BGH pA REGION 2612 3025 f1 origin REGION 3090 3414 SV40 promoter GENE 3432 4455 HygR REGION 4468 4840 SV40 pA REGION 5773 5100 C PUC origin GENE 6778 5918 C AmpR(complementary strand) SEQ ID NO: 3: pcDNA3.1-FVIII Molecule: 9975 bps DNA, circular REGION 1 655 CMV promoter GENE 783 3082 human FVIII domains A1 and A2 GENE 3083 3104 remainder of B-domain and add. nts GENE 3105 5162 human factor FVIII domains A3, C1, C2 REGION 5188 5402 BGH pA REGION 5465 5878 f1 origin REGION 5943 6267 SV40 promoter GENE 6285 7308 HygR REGION 7321 7693 SV40 pA REGION 8626 7953 C PUC origin GENE 9631 8771 C AmpR(complementary strand) SEQ ID NO: 4: pcDNA 3.1 sequence published by Invitrogen Molecule: 5597 bps DNA, circular REGION 209 863 CMV promoter REGION 895 1010 MCS REGION 1021 1235 BGH pA REGION 1298 1711 f1 origin REGION 1776 2100 SV40 promoter GENE 2118 3141 HygR REGION 3154 3526 SV40 pA REGION 4456 3786 C PUC origin GENE 5461 4601 C AmpR(complementary strand) SEQ ID NO: 5: pcDNA3.1Hygro(+)-zz, having 3 additonal nt “GGT” at position 4380 compared to SEQ ID NO: 4 Molecule: 5600 bps DNA, circular REGION 209 863 CMV promoter REGION 895 1010 MCS REGION 1021 1235 BGH pA REGION 1298 1711 f1 origin REGION 1776 2100 SV40 promoter GENE 2118 3141 HygR REGION 3154 3526 SV40 pA REGION 4459 3786 C PUC origin GENE 5464 4604 C AmpR(complementary strand) SEQ ID NO: 6: vector pTG1-A1AT SEQ ID NO: 7: vector pFGF8-hyg-s Molecule: 10705 bps DNA, circular REGION 1 586 CMV promoter GENE 676 2975 hFVIII domain A1 and A2 Gene 2976 2997 hinge remaining of B-domain and add. nts Gene 2998 5055 hFVIII domains A3, C1, C2 Region 5067 5916 polyA intron and polyA site from SV40 Gene 5928 7910 Hygromycin resistance Region 8346 8423 progesterone responsive element Gene 8681 9469 ampicillin resistance SEQ ID NO: 8: human wild-type factor VIII cDNA SEQ ID NO: 9: human wild-type factor VIII SEQ ID NOs: 10-12: linker peptides SEQ ID NO: 13: cDNA of hFVII a-form SEQ ID NO: 14: cDNA of hFVII b-form SEQ ID NO: 15: cDNA of human GCSF a-form, CDS: 41-661 SEQ ID NO: 16: cDNA of human GCSF b-form, CDS: 41-652 SEQ ID NO: 17: cDNA of human GCSF c-form, CDS: 229-828 SEQ ID NO: 18: cDNA of hvWF SEQ ID NOs: 19 and 20: G-CSF forward and reverse primer Start End Feature/Name SEQ ID NO: 21: vector PCR2.1d2-GCSFb Molecule: 4542 bps DNA; circular 293 907 GCSFb 1159 1596 f1 origin 1930 2724 KanR 2742 3602 AmpR 3747 4420 pUC origin SEQ ID NO: 22: vector pcDNA3.1-hyg(+)-GCSFb Molecule: 6237 bps DNA, circular 209 863 CMV promoter 970 1584 GCSFb 1658 1872 BGH pA 1935 2348 f1 origin 2413 2737 SV40 promoter 2755 3778 HygR 3791 4163 SV40 pA 5096 4423C PUC origin 6101 5241 C AmpR SEQ ID NO: 23: vector pCINeo-GCSFb Molecule: 6101 bps DNA, circular 1 1022 CMV promoter/intron 1106 1720 GCSFb 1796 2017 SV40 PolyA 2112 2567 f1 origin 2629 3047 SV40 enhancer/promoter 3092 3886 NeoR 3950 3998 PolyA 4409 5269 AmpR SEQ ID NO: 24: vector pCMVScript_GCSFb, Molecule: 4920 bps DNA, circular 1 602 CMV promoter 728 1342 GCSFb 1453 1836 SV40 PolyA 1974 2280 f1 origin 2449 2787 SV40 promoter 2822 3613 NeoR 3614 4063 HSV-TK PolyA SEQ ID NO: 25: pTG2-GCSFb-hyg-as Molecule: 6917 bps DNA, circular 5 591 CMV promoter 722 1336 GCSFb 1383 2228 SV40 intron + pA 2767 2255 C HSV TK PolyA 3782 2745 C HygR 4044 3796 C HSV TK promoter 4916 5704 AmpR SEQ ID NO: 26: cDNA of human GCSF b-form SEQ ID NO: 27: human GCSF b-form protein 

The invention claimed is:
 1. A method for preparing an immortalized human cell line stably transfected with a nucleic acid sequence comprising a gene encoding a human target protein or a derivative or mutant thereof, a promoter and a bovine growth hormone polyadenylation (polyA) signal, said promoter and polyA signal being linked to the 5′ and 3′ end of the gene encoding said human target protein, respectively, which method comprises transfecting an immortalized human host cell line under serum-free conditions with a transfection vector comprising said nucleic acid sequence and an origin of replication.
 2. The method of claim 1, wherein the human target protein is a human plasma protein selected from blood clotting factors such as selected from the group consisting of factor IX, factor VIII (wild-type and B-domain deleted), Factor VII/VIIa, and von Willebrand factor (vWF), growth factors, erythropoietin, colony-stimulating factors (CSFs), granulocyte stimulating factor (G-CSF), macrophage CSF (M-CSF), granulocyte-macrophage CSF (GM-CSF), cytokines, interleukins, protease inhibitors, alpha-1-antitrypsin (A1AT), chymotrypsin, transport proteins, hormones, inhibitory or regulatory acting proteins, and derivatives and mutants thereof.
 3. The method of claim 1, wherein in the transfection vector (i) the promoter is selected from viral promoters, housekeeping gene promoters and tissue specific promoters; and/or (ii) the origin of replication allows the replication and amplification of the plasmid in bacteria; (iii) the vector further carries at least one gene for a selection marker being under control of a promoter as defined in (i) above; and/or (iv) the vector further carries one or more further regulatory elements, said regulatory elements selected from splice sites, recombination sites, polyA sites, enhancers, multicloning sites and prokaryotic plasmid sequences.
 4. The method of claim 3 wherein the transfection vector carries a CMV promoter, a hygromycin gene, a polyA sequence and the gene of interest and is derived from the pcDNA3.1 vector having the sequence of SEQ ID NO:4 or
 5. 5. The method according to claim 1 wherein the immortalized human cell line (i) is capable of being transfected and being cultured under serum-free conditions; and/or (ii) has integrated adenoviral sequences into its genome; and/or (iii) is selected from the group of kidney, bladder, liver, lung, cardiac muscle, smooth muscle, ovary and gastrointestinal cells, preferably is a human kidney cell line; and/or (iv) is not capable of expressing a human prion protein.
 6. The method according to claim 5 wherein the kidney cells are human foetal kidney cells selected from 293 cells, 293T cells, or 293F cells.
 7. The method of claim 6, wherein the cell line is 293F and the transfection vector is derived from pcDNA3.1, wherein the vector encodes human blood clotting factor IX as shown by bps 939 to 2324 of SEQ ID NO:1, human A1AT as encoded by bps 973 to 2259 of SEQ ID NO:2, wt human factor VIII as shown in SEQ ID NO:9, or B-domain deleted human factor VIII as shown in SEQ ID NO:3, FVII/FVIIa, G-CSF including G-CSFb shown in SEQ ID NO:27, or von Willebrand factor.
 8. The method according to claim 1, wherein the serum-free transfection is effected in suspension culture without serum with a cationic transfection agent or calcium phosphate.
 9. The method according to claim 1, which further comprises selection for stably transfected cells by productivity, activity and product quality of the produced recombinant protein.
 10. The method according to claim 5 where the kidney cells are human foetal kidney cells 293F3 cells.
 11. The method according to claim 8, wherein the serum-free transfection is effected in suspension culture without serum with lipofectamine 2000 CD reagent.
 12. The method of claim 2 wherein the human target protein is selected from factor IX, factor VIII, wild-type factor VIII, B-domain deleted factor VIII, factor VII/VIIa, G-CSF, vWF and A1AT.
 13. The method of claim 2 wherein the human target protein is blood clotting factor IX as encoded by bps 939 to 2324 of SEQ ID NO:1, human A1AT as encoded by bps 973 to 2259 of SEQ ID NO:2, wt factor VIII as shown in SEQ ID NO:9, B-domain deleted human factor VIII as encoded by bps 783 to 5162 of SEQ ID N03, factor VII/VIIa as encoded by SEQ ID NOs:13 and 14, G-CSF as encoded by SEQ ID NOs:15, 16 and 17, or vWF as encoded by SEQ ID NO:18. 